Macrooxazoles A–D, New 2,5-Disubstituted Oxazole-4-Carboxylic Acid Derivatives from the Plant Pathogenic Fungus Phoma macrostoma

In our ongoing search for new bioactive fungal metabolites, four previously undescribed oxazole carboxylic acid derivatives (1–4) for which we proposed the trivial names macrooxazoles A–D together with two known tetramic acids (5–6) were isolated from the plant pathogenic fungus Phoma macrostoma. Their structures were elucidated based on high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy. The hitherto unclear structure of macrocidin Z (6) was also confirmed by its first total synthesis. The isolated compounds were evaluated for their antimicrobial activities against a panel of bacteria and fungi. Cytotoxic and anti-biofilm activities of the isolates are also reported herein. The new compound 3 exhibited weak-to-moderate antimicrobial activity as well as the known macrocidins 5 and 6. Only the mixture of compounds 2 and 4 (ratio 1:2) showed weak cytotoxic activity against the tested cancer cell lines with an IC50 of 23 µg/mL. Moreover, the new compounds 2 and 3, as well as the known compounds 5 and 6, interfered with the biofilm formation of Staphylococcus aureus, inhibiting 65%, 75%, 79%, and 76% of biofilm at 250 µg/mL, respectively. Compounds 5 and 6 also exhibited moderate activity against S. aureus preformed biofilm with the highest inhibition percentage of 75% and 73% at 250 µg/mL, respectively.

The molecular formula of compound 3 isolated from both supernatant and mycelia as a brown oil was established as C14H13NO4 (9 degrees of unsaturation) from the HR-ESIMS which showed an [M + H] + ion at m/z 260.0917 and an [M + Na] + ion at m/z 282.0737. Analysis of 1D and 2D NMR revealed a similar structure to 1 with the C-16 hydroxyl group missing in compound 3, but a double bond Δ 15−16 at δ 123.2 (C-15) and δ 121.0 (C-16) were recorded instead ( Table 3). The H-15 (δ 7.14) . Its NMR spectroscopic data displayed high similarities to those of compound 1, suggesting that they are close analogues. The only structural difference was the presence of the hydroxyl group at C-15 of compound 2 which was absent in compound 1. This was confirmed not only by the 1 H-1 H COSY coupling of H-15 (δ 5.33) to H-16a (δ 3.73)/H-16b (δ 3.78) but also by the HMBC correlation between H-15 (δ 5.33) and C-16 (δ 65.1). Interestingly, obtaining an optical rotation value approaching zero identified compound 2 to be a racemic mixture. Consequently, compound 2 was determined as a racemic mixture of methyl 5-(1,2-dihydroxyethyl)-2-(4-hydroxybenzyl)-oxazole-4-carboxylate, named macrooxazole B.
The molecular formula of compound 3 isolated from both supernatant and mycelia as a brown oil was established as C 14 H 13 NO 4 (9 degrees of unsaturation) from the HR-ESIMS which showed an [M + H] + ion at m/z 260.0917 and an [M + Na] + ion at m/z 282.0737. Analysis of 1D and 2D NMR revealed a similar structure to 1 with the C-16 hydroxyl group missing in compound 3, but a double bond ∆ 15−16 at δ 123.2 (C-15) and δ 121.0 (C-16) were recorded instead ( Table 3). The H-15 (δ 7.14) showed COSY correlations to H-16a (δ 5.58)/H-16b (δ 5.96) and HMBC correlations to C-5 (δ 155.6)/C-16 (δ 121.0) confirming the structure of the previously unreported metabolite 3 as methyl 2-(4-hydroxybenzyl)-vinyloxazole-4-carboxylate, named macrooxazole C. Fraction F1 (a mixture of compounds 2 and 4 (ratio 1:2)) was isolated as a yellow oil from both supernatant and mycelial extracts. On the basis of HR-ESIMS and 1D/2D NMR data of this mixture, the structure of compound 4 could be determined independently. HR-ESIMS data revealed the molecular formula of compound 4 as C 14  . Therefore, compound 4 was elucidated unambiguously as methyl 2-(4-hydroxybenzyl)-5-(oxiran-2-yl)-oxazole-4-carboxylate, named macrooxazole D. As can be seen in Figure 1, compounds 2 and 4 are both also present in the crude extract, suggesting they are both genuine natural products and that compound 2 does not only arise from macrooxazole D (4) as an isolation artefact during preparative HPLC separation. However, the conversion could already have taken place during fermentation of the fungus.
For an unambiguous confirmation of its structure, macrocidin Z (6) was synthesized starting by attaching 6-heptenoic acid (7) to the Evans auxiliary (R)-benzyl-2-oxazolidinone (Scheme 1) [18]. The resulting imide 8 was deprotonated at the α-position with NaHMDS to give an enolate which was quenched with iodomethane. The resulting 9.8:1 mixture of diastereomers was separated by column chromatography to afford the major isomer 9 in 79% yield. It was converted to the carboxylic acid 10 in 96% yield by adding LiOH and H 2 O 2 . The tetramic acid 12 was prepared according to a known protocol [17,19,20] by treatment of commercial Boc-Tyr(Allyl)-OH (11) with Meldrum's acid. Its acylation with carboxylic acid 10 via the two-step Yoshii-Yoda protocol [21,22] initially afforded 4-O-acyltetramate 13, which was rearranged to the 3-acyltetramic acid 14. A ring-closing metathesis reaction using Grubbs catalyst gave N-Boc-protected macrocidin Z 15 with an E-selectivity > 99% in 89% yield. Macrocidin Z (6) was obtained quantitatively upon removal of the Boc-protection group with TFA in 30% total yield over seven steps.

Biological Activities
The isolated metabolites were evaluated for their antimicrobial activity against various bacteria and fungi. The Minimum Inhibitory Concentration (MIC) values showed that only the new metabolite 3 as well as the known macrocidins 5 and 6 were active, whereas the remaining compounds were inactive against the organisms tested (See Table S1 in the supporting information). Macrocidin A (5) showed the strongest activity against Bacillus subtilis with an MIC value of 16.7 µg/mL which is the same value as that of oxytetracyclin used as positive control. The same compound 5 demonstrated weak activity against Mycobacterium smegmatis with an MIC value of 33.3 µg/mL. Compound 3 exhibited moderate activity against Mucor hiemalis with an MIC value of 66.7 µg/mL equal to that of nystatin used as a positive control. The latter also inhibited the growth of Bacilus subtilis at 66.7 µg/mL. Against Micrococcus luteus, compound 6 exhibited weak activity with an MIC value of 66.7 µg/mL. Furthermore, the ability of some of the isolated compounds to inhibit the proliferation of two mammalian cell lines including HeLa cells KB3.1 and mouse fibroblasts L929 was examined. Only the mixture of compounds 2 and 4 (ratio 1:2) showed weak cytotoxic activity against HeLa cells KB3.1 and mouse fibroblasts L929 with an IC50 value of 23 µg/mL for both cell lines, whereas compound 5 and 6 only showed a slight inhibition of HeLa cells KB3.1 proliferation (See Table S2 in the supporting information).
Moreover, the isolated pure compounds except compound 4 (which was not tested because it was isolated as a mixture) were evaluated for their effectiveness in inhibiting biofilm formation and preformed biofilm of Staphylococcus aureus ( Table 4). The new compounds 2 and 3 showed moderateto-weak activity against biofilm formation, with respective inhibition percentages of 65% and 75% at the highest concentration of 250 µg/mL. Compounds 5 and 6 inhibited 61% and 19% of the bacterial biofilm at 15.6 µg/mL, respectively ( Figure 5). Interestingly, the test compounds also displayed activity against preformed biofilm of S. aureus as represented in Table 4 below. Macrocidins A (5) and Z (6) exhibited moderate activity against preformed biofilm of S. aureus with the highest percentage of inhibition of 75% and 73% at 250 µg/mL, respectively.

Biological Activities
The isolated metabolites were evaluated for their antimicrobial activity against various bacteria and fungi. The Minimum Inhibitory Concentration (MIC) values showed that only the new metabolite 3 as well as the known macrocidins 5 and 6 were active, whereas the remaining compounds were inactive against the organisms tested (See Table S1 in the supporting information). Macrocidin A (5) showed the strongest activity against Bacillus subtilis with an MIC value of 16.7 µg/mL which is the same value as that of oxytetracyclin used as positive control. The same compound 5 demonstrated weak activity against Mycobacterium smegmatis with an MIC value of 33.3 µg/mL. Compound 3 exhibited moderate activity against Mucor hiemalis with an MIC value of 66.7 µg/mL equal to that of nystatin used as a positive control. The latter also inhibited the growth of Bacilus subtilis at 66.7 µg/mL. Against Micrococcus luteus, compound 6 exhibited weak activity with an MIC value of 66.7 µg/mL. Furthermore, the ability of some of the isolated compounds to inhibit the proliferation of two mammalian cell lines including HeLa cells KB3.1 and mouse fibroblasts L929 was examined. Only the mixture of compounds 2 and 4 (ratio 1:2) showed weak cytotoxic activity against HeLa cells KB3.1 and mouse fibroblasts L929 with an IC 50 value of 23 µg/mL for both cell lines, whereas compound 5 and 6 only showed a slight inhibition of HeLa cells KB3.1 proliferation (See Table S2 in the supporting information).
Moreover, the isolated pure compounds except compound 4 (which was not tested because it was isolated as a mixture) were evaluated for their effectiveness in inhibiting biofilm formation and preformed biofilm of Staphylococcus aureus ( Table 4). The new compounds 2 and 3 showed moderate-to-weak activity against biofilm formation, with respective inhibition percentages of 65% and 75% at the highest concentration of 250 µg/mL. Compounds 5 and 6 inhibited 61% and 19% of the bacterial biofilm at 15.6 µg/mL, respectively ( Figure 5). Interestingly, the test compounds also displayed activity against preformed biofilm of S. aureus as represented in Table 4 below. Macrocidins A (5) and Z (6) exhibited moderate activity against preformed biofilm of S. aureus with the highest percentage of inhibition of 75% and 73% at 250 µg/mL, respectively.  Apart from the strong herbicidal activity of macrocidins [15,16], no other activity has been reported for this class of compounds as far as we know. The current paper therefore constitutes the first extensive evaluation of the biological effects for this class of compounds.

Fungal Material
The fungal strain Phoma macrostoma DAOMC 175,940 was originally isolated from the Canadian thistle Circium arvense collected in Quebec, Canada in 1979. It constitutes one of the original producer strains of the macrocidins [15,16,23] and was kindly provided by the CCFC (Canadian Collection of Fungal Cultures, Ottawa, ON, Canada).

Small-Scale Fermentation and Extraction
The fungus was cultivated in Q6 1 2 medium (10 g/mL glycerol, 2.5 g/mL d-glucose, 5 g/mL cotton seed flour and pH = 7.2) [24]. A well-grown culture from a yeast-malt (YM) agar plate (10 g/mL malt extract, 4 g/mL yeast extract, 4 g/mL D-glucose, 1.5% agar and pH = 6.3) was cut into small pieces using a cork borer (7mm), and eight pieces were inoculated into 6 × 500 mL Erlenmeyer flasks, each containing 200 mL of the Q6 1 2 medium. The culture was incubated at 23 • C on a rotary shaker (140 rpm). The growth of the fungus was monitored by measuring the amount of free glucose using Diastix Harnzuckerstreifen (Bayer). After glucose depletion, small samples were taken to monitor secondary metabolite production over a period of 14 days (searching for the mass spectra and UV/Vis spectra that were reported to be typical for the macrocidins) and a stagnation of the titres of the putative macrocidin derivatives was observed by HPLC-MS between 8 and 14 days.
Then, the fermentation was terminated and the supernatant and mycelia were separated by filtration. The supernatant was extracted with equal amount of ethyl acetate (200 mL) and filtered through anhydrous sodium sulphate. The resulting ethyl acetate extract was evaporated to dryness by means of rotary evaporator. The mycelia was extracted with 200 mL of acetone in an ultrasonic bath at 40 • C for 40 min, filtered and the filtrate evaporated. The remaining water phase was suspended in equal amount of distilled water and subjected to same procedure as the supernatant.

Scale Up of Production in Shake Flask Batches and Extraction
Four well-grown 17-day-old YM agar plates of the mycelial culture were cut into small pieces using a 7 mm cork borer and 8 pieces inoculated in 30 × 500 mL Erlenmeyer flasks containing 200 mL of Q6 1 2 medium. The culture was incubated at 23 • C on a rotary shaker (140 rpm) for 13 days. Fermentation was aborted 10 days after the depletion of free glucose.
The mycelia and supernatant from the batch fermentation were separated via filtration. The mycelia was extracted with 4 × 500 mL of acetone in an ultrasonic water bath at 40 • C for 40 min. The extracts were combined and the solvent evaporated by means of a rotary evaporator. The remaining water phase was four times subjected to the same procedure as mycelium in small-scale extraction yielding 949 mg dark brown crude extract. The supernatant (6 L) was extracted with an equal amount of ethyl acetate and filtered through anhydrous sodium sulphate. The resulting ethyl acetate extract was evaporated to dryness by means of rotary evaporator to afford 238 mg of brown crude extract.
A total of 5 mg of F1 was further purified by reversed phase HPLC (solvent A (H 2 O + 0.1% FA)/solvent B (ACN + 0.1% FA)), elution gradient 20-50% solvent B for 35 min followed by maintaining isocratic condition at 100% solvent B for 5 min with a preparative HPLC column (VP Nucleodur 100-10 C18ec column (250 × 10 mm, 7 µm: Machery-Nagel, Düren, Germany) as stationary phase) and a flow rate of 8 mL/min, to afford only compound 2. The absence of the peak of compound 4 in the obtained HPLC chromatogram suggests the instability of compound 4 which easily turns into compound 2.  Table 2.  Table 2.  Table 3.

Conclusions
During the course of our search for new biologically active secondary metabolites, four previously undescribed oxazole carboxylic acid derivatives were isolated from the plant pathogenic fungus Phoma macrostoma. As far as we know, these metabolites constitute the first series of oxazole derivatives isolated from this genus. Investigation of the antimicrobial activity of the new isolates revealed that only compound 3 displayed moderate activity against Bacillus subtilis and Mucor hiemalis. Although none of the isolates displayed any antibacterial activity against S. aureus, compounds 2 and 3 showed moderate to weak inhibition of biofilm formation and preformed biofilm of the bacteria. Moreover, two known tetramic acids macrocidins, A and Z, were also characterized. The so far unclear structure of macrocidin Z was confirmed in this study by its first total synthesis. Even though the macrocidins are well known for displaying a strong herbicidal activity, their biological effects have also been extensively evaluated in the present work and they turned out to possess an interesting antibiofilm effect against S. aureus. Thanks to their ability to inhibit biofilm formation, these are likely to be considered as promising candidates for the development of lead molecules that could function as adjunctive agents in combination therapy with antibiotics.